Virus detection method, primers therefor and screening kit

ABSTRACT

Discloses a novel detection and typing method for viruses, such as human papillomaviruses, based on real-time PCR using self-probing amplicon fluorescent primers. The method comprises: (IA) contacting the sample with a self-probing amplicon (‘virus self-probing amplicon’) comprising (i) a virus primer capable of hybridising to at least one target viral nucleic acid sequence and undergoing amplification thereof under primer amplification conditions to form a virus primer extension product; (ii) a virus probe comprising a nucleic acid sequence complementary to a target sequence of the virus primer extension product and capable of hybridisation thereto, provided that the self-probing amplicon is adapted to ensure that the virus probe is unresponsive to amplification under the primer amplification conditions; and (iii) a member of a virus signalling system, which system is capable of causing a detectable signal to be effected on hybridisation of the virus probe sequence to the virus primer extension product, whereby presence or absence of the target viral nucleic acid sequence in the sample is indicated by the detectable signal; (IB) amplifying the product of step (IA) under the primer amplification conditions to an extent enabling the detectable signal to be effected after step(II); and (II) separating the virus primer extension product from the target viral nucleic acid sequence; allowing the virus probe to hybridise to the target sequence of the virus primer extension product; and monitoring the signalling system. This method is quick, simple, specific, sensitive, and capable of estimating viral load per cell. The results of over 100 HPV typing reactions performed on cell lines, biopsies and cervical cytobrush samples are given which, when compared to the current reference HPV detection and typing technique, present a kappa value of 0.89. The method is also applicable to other viruses, such as SV40.

The present invention relates to an improved method for detecting,typing and characterising the presence of viruses, particularly thosethat have been associated with carcinogenic activity in mammals, such ashuman papillomavirus and SV40, and to primers and probes for use in themethod. The invention further relates to a diagnostic kit and screeningmethod, which uses the kit.

As well as there being various viruses that have been associated withcarcinogenic effects and therefore there being a need to be able toidentify the presence of such viruses in clinical samples, it is alsoimportant to be able accurately to determine the viral load per cell andintegration status of the virus in the affected organism (mostimportantly, in humans). Accordingly, it is desired to provide means forestablishing the infecting virus type, the viral load (per cell ie thenumber of viral copies per cell or ratio of viral genome:human genome)and the integration state (ie whether the viral DNA is ‘free’ in thecytosol of the cell or integrated into the host genome) of the virus,since these factors are known to have profound implications for patientprognosis.

These issues will first be illustrated with reference to two particularvirus categories: human papillomaviruses (HPV) and SV40.

Cervical cancer is the second most frequent cause of death from cancerin women, worldwide. Cervical screening programmes reduce the incidenceof cervical cancer; however, 50% of invasive cervical cancers arise inwomen screened using existing cytological methodologies. In recentyears, it has been established that a subset of human papillomaviruses(HPV) are associated with cervical cancer, and it is estimated that HPVDNA is present in over 99% of these cancers. There are currently 84types of HPV, around 30 of which infect the genital tract. Therefore,HPV detection and typing techniques have been proposed as an adjunct to,or replacement for, the current cytological screening regime. Clearly,the success of such strategies will depend on the development of rapid,reliable, sensitive and specific HPV-detection methods applicable in theclinical setting.

Currently, there are eight main approaches to the detection and typingof HPV, all of which display advantages and disadvantages, depending onapplication. These are summarised in Table 1, from which it can be seenthat no single technique performs optimally in both clinical andresearch settings. By way of background, the PCR method has beenintroduced as the most sensitive method for the detection of HPV DNA inclinical specimens. However, a significant heterogeneity at thenucleotide level is found between the different HPV genotypes. This hashampered the development of a simple, universal PCR test for thedetection of all HPV genotypes. Despite this, HPV PCR methods have beendeveloped, allowing the detection of a broad spectrum of mainlymucosotropic HPV genotypes. TABLE 1 Current HPV Detection and TypingTechniques. Analysis Comments Technique Methodology AdvantagesDisadvantages Reference PCR-based Consensus Primers Enzyme-LinkedAccurate, Laborious, Jacobs et al J Clin Immunosorbent Assay sensitiveexpensive. Microbiol. 35 791-5 (1997) Restriction Fragment Quick, cheapNon-quantitative, Rodu et al in Length Polymorphism low sensitivity.Biotechniques 10 632-7 (1991) Dot blot Quick, accurate Limitedresolution, Gravitt et al J Clin non-quantitative. Microbiol 36 3020-7(1998) Sequencing Accurate, Laborius, expensive. Vernon et al J Clinsensitive Multiple types Microbiol 38 651-5 problematic (2000) Taqman ™Accurate, Complex, expensive. Swan et al J Clin probes sensitive,Microbiol 35 886-91 quantitative (1997) Type- Agarose gel Quick, cheapNon-quantitative, Evander et al in Arch specific electrophoresis lowsensitivity. Virol 116 221-33 (1991) primers Non-PCR based Hybrid EnzymeLinked Semi-quantitative, Low sensitivity, Vernon et al J Clin Capture ™Immunosorbent commercially specificity and Microbial 38 651-5 Assayavailable resolution. (2000)

HPV is genetically composed of early (E) and late (L) genes, which arefunctionally divided into several open reading frames (ORFs): viralreplication (E1), regulation of transcription (E2), coding forcytoplasmic proteins (E4) and malignant transformation (E5, E6, E7) asearly genes, and coding for capsid proteins (L1, L2) as late genes.

A combination of the general primers GP5 and GP6, originally selectedfrom the HPV L1 region on the basis of sequence information from HPV-6,-11, -16, -18, -31 and -33, was found to amplify target DNA of at least27 mucosotropic HPV genotypes under conditions that allow mismatchacceptance. The general primers GP5 (5′-TTT GTT ACT GTG GTA GAT AC-3′)and GP6 (3′-ACT AAA TGT CAA ATA AAA AG-5′) (Snijders etal, J. Gen.Virol. 71 173-181 (1990)), which span a region of 140-150 bp from the L1open reading frame of a broad spectrum of HPV genotypes, were used ingeneral primer-mediated PCR. The strength of this (GP-PCR) method hasbeen further substantiated by the detection of HPV DNA in 100% ofcervical scrapes cytomorphologically classified as Pap IV (carcinoma insitu) and Pap V (carcinoma) in the Netherlands. This suggests that, inthe Dutch population, all genital high risk HPV's can be detected bythis assay.

But, using GP-PCR in routine diagnostic practice, it has been found thata small number of clinical samples give rise to ambiguous results,reflected by weak GP-PCR signals accompanied by a relatively highbackground of co-amplified cellular DNA. It was then found thatincreased primer length contributes to more efficient amplification, andso the GP5 and GP6 primers were elongated with highly conservedsequences at the 3′ ends. When the use of these GP5+ and GP6+ primers inthe PCR was compared with the original GP5 and GP6 PCR on 1 ng clonedDNA of various HPV types, it was found that the elongated generalprimers were significantly more sensitive.

In order to facilitate PCR-based HPV detection and typing, acolorimetric microtitre plate based hybridisation assay was developed.The method used one biotinylated primer (bio-GP6+) in the GP-PCR.Biotinylated PCR products were captured on streptavidin-coatedmicrotitre plates, denaturated and hybridised to digoxigenin-(DIG-)labelled HPV-specific internal oligonucleotide probes. The DIG-labelledhybrids were detected using an enzyme immunoassay (EIA). In adevelopment of this technique, two cocktails of DIG-labelled HPVtype-specific oligonucleotide probes and an EIA have been used as abasis to develop a group-specific detection method for 14 high-risk(types 16, 18, 31, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) and 6low-risk (types 6, 11, 40, 42, 43 and 44) HPVs following a generalprimer GP5+/bioGP6+-mediated PCR. This technique is hereinafter referredto as PCR-EIA. The sensitivity of this high-risk/low risk (HR/LR) HPVPCR-EIA ranged from 10 to 200 HPV copies, depending on the HPV type.

Some fluorogenic probe assays for HPV DNAs are based on the PCRamplification of a portion of the L1 open reading frames of HPV-16, -18,-31, -33, and -35 DNAs by using genotype-specific probes that bind tothe amplified DNA. In the TaqMan assay, the probes are blocked at their3′ termini and hence cannot be extended by the polymerase. If, duringthe course of primer extension, Taq polymerase encounters a bound probe,its 5′→3′ exonuclease activity degrades the probe, releasing the 5′fluor from the 3′ quencher. This causes an increase in the fluorescenceemitted by the reporter which, in the presence of an excess amount ofthe probe, is directly related to the amount of HPV DNA present in thesample before amplification. ‘Molecular beacons’ comprise a probeflanked by a hairpin loop that holds a fluorophore and quencher in closeproximity until specific binding of the probe to its target opens outthe structure, producing a fluorescent signal.

In general, HPV DNA is present as an episomal form in cervicalintra-epithelial neoplasia (CIN) lesions, and the principal form of theviral DNA in invasive cancers is integrated into the host genome. Inmost cervical carcinomas, HPV genomes are integrated into host cellchromosomes and transcribed into mRNAs encompassing viral and cellularsequences. In contrast, in early pre-neoplastic lesions, HPV genomespersist as episomes, and derived transcripts contain exclusively viralsequences. Thus, detection of HPV transcripts derived from integratedHPV genomes may specifically indicate both CIN lesions at high risk forprogression as well as invasive cervical cancers.

When the HPV DNA becomes integrated into the cellular chromosome,generally some part of the E2 ORF is lost, releasing suppression of E6and E7 mRNA expression. The E6 and E7 proteins can activate someoncogenes and/or inactivate host tumour suppressor gene products (p53,pRB), resulting in uncontrolled cellular growth and malignanttransformation.

Integration of HPV DNA is therefore considered an important geneticchange in the process of cervical carcinogenesis, and being able todetermine the level of integration is believed to be important,diagnostically. Some detection techniques therefore focus on identifyingand quantifying disruptions in the early genes, particularly E1/E2 andE6/E7. These have been carried out using techniques such as multiplexPCR (where primers for each sequence are included in the same reaction)and RT-PCR that allows discrimination of HPV mRNAs derived fromintegrated and episomal viral genomes.

Hitherto-known techniques therefore require a plurality of techniquesand/or probes to detect HPV types and integration status, which istime-consuming, slow and generally unsatisfactory for clinical use.Furthermore, none of these techniques satisfactorily deal with thedetermination of viral load, since none result in a measure of thenumber of viral copies per cell. These systems have used differentmethods for the quantitation of virus copy number and cell copy numberand thus the two are not strictly comparable. Previous methodsincorporating assays for the β-globin or other housekeeping genes of thegenomic DNA have, at most, given rise to a range of viral DNA copies permicrogram of cellular DNA, rather than per cell. Other uses ofhousekeeping genes have been as PCR controls or to determine PCR limitsand not to provide information about viral load.

Another disease caused by HPV is Recurrent Respiratory Papillomatosis(RRP), which is the most common benign tumour of the larynx, and is aserious condition with no satisfactory treatment and around 2-7%malignant transformation. RRP is caused by one of two HP viruses,namely, HPV-6 and HPV-11. However, these viruses can also be detected inthe oropharynx of over 50% of normal school-age children and so hostfactors are clearly important.

It is postulated that HPV transmission is mediated during transitthrough the birth canal in juvenile onset RRP and during oro-genitalcontact in adult onset RRP. Patients with the disease have no obviousimmune deficit.

Another example of a virus that has been linked to cancer, particularlymesotheliomas (cancers affecting the mesothelial cells in the lining ofthe chest and lung) is Simian virus 40 (SV40), named after its origin inthe Simian monkey. It was discovered as a contaminant in early doses ofpolio vaccine and was initially thought to be harmless in humans. It wasfound to cause tumours in laboratory animals and since then has beenlinked to osteosarcomas, pituitary, thyroid, brain and neurologicaltumours (eg glioblastomas, astrocytomas, ependymomas and pappillomas ofthe choroids plexus). SV40 is now believed to be more tumorigenic thatnHPV, since one copy of SV40 per cell is believed to be capable oftransforming the cell. Nevertheless, it is understood that mesotheliomasassociated with SV40 can take up to 20 to 40 years to develop, but isvery pernicious, causing death in about 18 months, with about 3,000victims per annum in the USA alone.

The highly tumorigenic nature of SV40 is attributed to the fact that itproduces very small amounts of ‘large T-antigen’, which can knock outboth the p53 and Rbs regulatory pathways simultaneously, whereas HPV hasto produce two agents (E6 and E7) to do so. SV40 also damageschromosomes by re-arrangement of DNA. Accordingly, there is a need for amethod that can accurately detect, type and quantify (load per cell andintegration status) of SV40, too.

The PCR technique originally used for the detection of SV40-likesequences in ependymoma and choroids plexus tumours has become theprinciple method used by the majority of workers in the field to studythe presence of SV40 in human pleural mesothelioma. This methodcomprises extraction of the DNA from a sample of the tumour, and PCRamplification thereof with primers designed both to check the quality ofthe extracted DNA and then the presence in it of any SV40 DNA-likesequences.

The primers (SV.for 3/SV.rev) most frequently used have been designed toamplify the sequence that codes for Rb, p107 and Rb2/p130 bindingdomains of the large T-antigen. The incidence rates of SV40 detectionusing this technique in the hands of various workers range from 0% to100% (see Jasani et al in Fron in Biosci 6 e12-22 (2001)), which may atleast in part be due to the varying efficiency of the DNA extraction andPCR amplification and product detection methods.

In terms of detection of viral load, there have also been mixed results,ranging from 1 to 100 copies of SV40 genome copies per PCR reaction, andit is unclear whether the lower end of this range includes viral copiesper cell rather than per PCR reaction in all cases. An improvement insensitivity has been found when using Southern blotting based on probestargeted at SV40 T antigen sequences. Several researchers have usedmultiple primer sets to identify genomic sequences from different partsof the genome, as well as sequence-specific variation. Despite this,doubts have persisted about the authenticity of SV40 DNA detected inhuman tissues, and it has proved necessary to organise amulti-institutional study involving nine laboratories under the auspicesof the FDA in the USA.

Accordingly, the present invention provides a new method for virustyping that is referred to hereinbelow as “Viral Evaluation usingSelf-Probing Amplicons” (VESPA). VESPA is a real-time PCR-basedtechnique that uses self-probing amplicon primers, which are describedby Whitcombe et al in Nat Biotechnol 17 804-7 (1999).

Such self-probing amplicon primers comprise a specific probe sequencethat is held in a hairpin loop configuration by complementary stemsequences on the 5′ and 3′ sides of the probe. A fluorophore (such as6-carboxyfluorescein) attached to the 5′-end is quenched by a moiety(such as methyl red) joined to the 3′-end of the loop. The hairpin loopis linked to the 5′-end of a primer via a PCR stopper or blocker. Afterextension of the primer during PCR amplification, the specific probesequence is able to bind to its complement within the same strand ofDNA. This hybridisation event opens the hairpin loop, so thatfluorescence is no longer quenched and an increase in signal isobserved. The PCR stopper on the primer prevents read-through to theprobe, which could lead to opening of the hairpin loop in the absence ofthe specific target sequence. Such read-through would lead to thedetection of non-specific PCR products, eg primer dimers or mis-primingevents.

Accordingly, as used herein, the term “self-probing amplicon” refers toa molecule comprising a primer component, a probe component and asignalling system (which may comprise the fluorophore/quencher system asdescribed above or an alternative), as described above and by Whitcombe(1999) (q.v.). Thus, unlike ‘molecular beacons’ and TaqMan probes thathave been used, the self-probing amplicon system does not require aseparate probe. The self-probing amplicons work in a unimolecularmanner, leading to advantages both in terms of simplicity and signal tonoise ratios when compared to the bi-molecular probing of ‘molecularbeacons’ and TaqMan. Furthermore, such self-probing amplicons can beadapted to mutation or allelic discrimination by monitoring thefluorescence at a temperature where the probe has dissociated from atarget with a mismatch but remains bound to a complementary target. Thisis different from the allelic discrimination by self-probing ampliconsdescribed by Whitcombe et al, as they used the ARMS system, whereby theprimer rather than the probe is sited over the polymorphic site.

VESPA methodology is well suited to virus detection since it is simpleto perform rapid, highly specific, sensitive, reproducible, and has thepotential to measure viral load. The method of the invention has beentested and it has produced typing results on 108 samples, including celllines, cervical cytobrush samples and tumour biopsies, and preliminaryviral load per cell data on 16 clinically-defined samples has beenobtained, as described hereinbelow in the Examples.

Furthermore, when compared to both prior art front-line virus-detectionand typing methodologies, Hybrid Capture II™ and PCR-EIA, VESPA appearstechnically less demanding and able to produce results more rapidly. Thesensitivity of VESPA in cell lines is at least two orders of magnitudebetter than that reported by Digene (5000 copies) for Hybrid Capture II™(from Digene Corporation, Silver Spring, Md.), which has been approvedby The Federal Drugs Administration (FDA in the USA) for HPV screening.VESPA has comparable sensitivity to other previously-published HPVdetection techniques, including PCR-EIA (ie in the range of from 1 to100 viral copies), in which the detection limit is calculated usingenriched control targets against a low background of genomic DNA. Theexact viral threshold for immediate risk of carcinogenesis iscontroversial (and may vary with HPV type and patient), but is likely tobe well above VESPA's lower detection limit.

The presence of inhibitors (eg haemoglobin) that may prevent efficientDNA amplification in cervical samples has been previously reported.Using a standard chloroform extraction based method of DNA purification,we have demonstrated that it is possible to improve the signal producedby VESPA to detect HPV in cervical smear samples only weakly positivewhen purified by the freeze-thaw technique usually used with PCR-EIA.VESPA is type-specific and compares favourably with Hybrid Capture™, inwhich samples are only categorised into “Low Risk” and “High Risk” HPVtypes. The HPV typing achieved using VESPA correlates well in our handswith data obtained using PCR-EIA (κ=0.89), the most established HPVtyping method.

In order to eliminate the false positives often associated with nestedPCR, such as in the case of SV40, an assay based on highly sensitivefirst round amplification, in order to detect very low viral copynumbers, and highly specific second round amplification, may be used.The primer sites used in the first round of amplification may based onknown and established primer sites. Preferably, for SV40, all primersand probes are capable of targetting the large T antigen site in eachvirus, such that each assay is equivalent. In the second step, usingself-probing amplicon primers and real-time PCR, amplification anddetection take place concurrently.

As well as detection and typing, VESPA has the potential to estimateviral load (ie per cell rather than gross amount of virus present).There is increasing evidence that viral load per cell is a criticaldeterminant in patient prognosis. Indeed, the so-called “high risk”types may not be more potent due to the increased oncogenicity of theirtransforming proteins, for example, but simply because they proliferatemore efficiently, overwhelming the immune response.

In order to expand the capability of VESPA for use in virus screening,the present invention further provides a degenerate virus self-probingamplicon mix for use in conjunction with a tailed general primer. Byusing a tailed primer, it is possible to introduce a consensus site thatenables a single self-probing amplicon to recognise many different virusamplification products. Theoretically, this primer combination candetect over forty different HPV types.

In the PCR-EIA technique described by Jacobs et al in J Clin Microbiol35, 791-5 (1997), viral DNA is amplified using the prior art consensusprimers (GP5+/GP6+), then analysed by ethidium bromide staining ofelectrophoresed agarose gels. If an amplification product is observed,then it is typed by ELISA using type-specific probes. In practice,however, all samples are typed by ELISA, since agarose gelelectrophoresis is not sensitive enough to detect poorly amplified DNA.VESPA could circumvent this problem by providing improved sensitivity atthe pre-typing stage. Although the concept of a tailed primer is known,the use of the tail as a primer site and the primer as a probe-bindingsite for a self-probing amplicon is new.

Therefore, the method of the invention for the characterisation ofviruses such as human papillomavirus or SV40 infection is quicker (<1hour); more specific (single base discrimination); and less laborious(single step) than currently available techniques and, unlike mosttechniques, is capable of estimating viral load per cell. It can also beused to determine integration status of the virus. Especially importantis the ability of the technique of the invention to determine aplurality of virus types by using tailed primers.

The invention is further described below, with reference to thefollowing Figures:

FIG. 1: relates to HPV-16 detection by the method of the invention, inwhich:

FIG. 1 a shows the results of HPV typing reactions using self-probingamplicon primers specific for HPV-16 (Sc16), a positive control (HeLafor HPV-18); a negative control (no DNA); and DNA extracted from a HPV16 specific cell line (Caski); and

FIG. 1 b shows the results of HPV typing reactions using theself-probing amplicon primers specific for HPV-18 (Sc18); a positivecontrol (Caski for HPV-16), a negative control (no DNA); and DNAextracted from an HPV-18 containing cell line (HeLa).

FIG. 2 relates to examples of positive traces produced by VESPA andshows the results of HPV typing experiments using clinical samplespreviously typed using PCR-EIA. Primers specific for HPV-6 (Sc6) areshown in FIG. 2 a, HPV-11 (Sc11) in FIG. 2 b, HPV-16 (Sc16) in 2 c,HPV-18 (Sc18) in 2 d, HPV-31 (Sc31) in 2 e, HPV-33 (Sc33) in 2 f, HPV-39(Sc39) in 2 g, and HPV-51 (Sc51) in 2 h.

FIG. 3 demonstrates the quantitative nature of VESPA for HPV-16. Itshows an HPV-16 dilution series using Sc16. A dilution series of SiHacells was made from 50,000 cells per reaction to 1 cell per reaction.

FIG. 4 demonstrates the quantitative nature of VESPA for humanbeta-globin using ScBG and the dilution series as for FIG. 3.

FIG. 5 is a graph showing estimation of viral load using VESPA.

FIG. 6 is a diagrammatic representation of a strategy for virus, such asdegenerate HPV, detection using VESPA.

FIG. 7 demonstrates the ability of the degenerate self-probing ampliconmix to detect HPV types-6, -16 and -18.

FIGS. 8 and 9 are schematic representations of virus, such as HPV,typing using self-probing amplicons, in which FIG. 8 is a diagram of aself-probing amplicon and FIG. 9 shows the extension and binding stagesof the self-probing amplicon with respect to the viral DNA.

FIG. 10 relates to results obtained in applying the VESPA technique toSV40. Shown in FIG. 10 a-c are LightCycler fluorescence profilesproduced by ScSV40, ScJC and ScBK, respectively. Each self-probingamplicon (Sc) is shown challenged with pre-amplified Ori-3 (SV40), Mad 1(JC), X (BK) and sample DNA. Shown in FIG. 10 d-f are iCyclerfluorescence profiles produced under the same conditions.

FIG. 11 is a schematic representation of a strategy for determiningintegration state of ca virus, using the method according to theinvention.

The present invention, therefore, provides a method for one or more of:

(a) detection;

(b) typing;

(c) determination of viral load per cell; and/or

(d) determination of the integration state of an animal, including amammalian, virus in a sample from an animal, including a mammal,suspected of comprising one or more target viral nucleic acidsequence(s),

which method comprises:

(IA) contacting the sample with a self-probing amplicon (‘virusself-probing amplicon’) comprising

-   -   (i) a virus primer capable of hybridising to at least one target        viral nucleic acid sequence and undergoing amplification thereof        under primer amplification conditions to form a virus primer        extension product;    -   (ii) a virus probe comprising a nucleic acid sequence        complementary to a target sequence of the virus primer extension        product and capable of hybridisation thereto, provided that the        self-probing amplicon is adapted to ensure that the virus probe        is unresponsive to amplification under the primer amplification        conditions; and    -   (iii) a member of a virus signalling system, which system is        capable of causing a detectable signal to be effected on        hybridisation of the virus probe sequence to the virus primer        extension product, whereby presence or absence of the target        viral nucleic acid sequence in the sample is indicated by the        detectable signal;

(IB) amplifying the product of step (IA) under the primer amplificationconditions to an extent enabling the detectable signal to be effectedafter step (II); and

(II) separating the virus primer extension product from the target viralnucleic acid sequence; allowing the virus probe to hybridise to thetarget sequence of the virus primer extension product; and monitoringthe signalling system.

(FIGS. 8 and 9 illustrate the method, schematically.)

Particularly preferred is a method according to this invention, whereinthe nucleic acid sequence(s) is/are DNA sequence(s).

In order to be particularly effective in determining viral load percell, the method is preferably carried out using a self-probing amplicondesigned to detect DNA of a cell housekeeping gene. In the context ofthis invention, “cell housekeeping” or “housekeeping” gene refers to agene that is stably present in the cell and therefore suitable foracting as a baseline indicator for the presence of the cell in thesample.

Accordingly, the present invention further provides a method for one ormore of:

(a) detection;

(b) typing;

(c) determination of viral load per cell; and/or

(d) determination of the integration state of a virus in a samplesuspected of comprising one or more target viral nucleic acidsequence(s),

which method comprises:

(IA) contacting the sample with a self-probing amplicon (‘virusself-probing amplicon’) comprising

-   -   (i) a virus primer capable of hybridising to at least one target        viral nucleic acid sequence and undergoing amplification thereof        under primer amplification conditions to form a virus primer        extension product;    -   (ii) a virus probe comprising a nucleic acid sequence        complementary to a target sequence of the virus primer extension        product and capable of hybridisation thereto, provided that the        self-probing amplicon is adapted to ensure that the virus probe        is unresponsive to amplification under the primer amplification        conditions; and    -   (iii) a member of a virus signalling system, which system is        capable of causing a detectable signal to be effected on        hybridisation of the virus probe sequence to the virus primer        extension product, whereby presence or absence of the target        viral nucleic acid sequence in the sample is indicated by the        detectable signal; and

(IB) amplifying the product of step (IA) under the primer amplificationconditions to an extent enabling the detectable signal to be effectedafter step (II);

(II) separating the virus primer extension product from the target viralnucleic acid sequence; allowing the probe to hybridise to the targetsequence of the virus primer extension product; and monitoring thesignalling system;

(IIIA) contacting a housekeeping nucleic acid sequence from the samplewith a self-probing amplicon (‘housekeeping self-probing amplicon’)comprising

-   -   (i) a housekeeping primer capable of hybridising to the        housekeeping nucleic acid sequence and undergoing amplification        thereof under primer amplification conditions to form a        housekeeping primer extension product;    -   (ii) a housekeeping probe comprising a nucleic acid sequence        complementary to a target sequence of the housekeeping primer        extension product and capable of hybridisation thereto, provided        that the housekeeping self-probing amplicon is adapted to ensure        that the probe is unresponsive to amplification under the primer        amplification conditions; and    -   (iii) a member of a housekeeping signalling system, which system        is capable of causing a detectable signal to be effected on        hybridisation of the housekeeping probe sequence to the        housekeeping primer extension product, whereby presence or        absence of the target housekeeping nucleic acid sequence in the        sample is indicated by the detectable signal;

(IIIB) amplifying the product of step (IIIA) under the primeramplification conditions to an extent enabling the detectable signal tobe effected after step (IV); and

(IV) separating the housekeeping primer extension product from thehousekeeping nucleic acid sequence; allowing the housekeeping probe tohybridise to the target sequence of the housekeeping primer extensionproduct; and monitoring the housekeeping signalling system.

Suitable housekeeping genes include β-globin, actin, tropomyosin andglyceraldehyde phosphate dehydrogenase (GAPDH). Preferably, thehousekeeping gene is β-globin, which is particularly suitable for actingas a human genomic DNA reference gene in the viral load per cellanalysis according to this invention.

Suitably, viral load per cell can be determined by comparing the signalseffected on hybridisation of, on one hand, the ‘viral self-probingamplicon’ and, on the other hand, the ‘housekeeping self-probingamplicon’. For example, viral load per cell can be quantified as asimple ratio of the two signals. (FIGS. 4 and 5 refer.)

Conveniently, for determining both viral type(s) and load per cell inthe same experiment, a plurality of signalling systems may be employed,whereby the signals distinguishably identify presence of virus type andhousekeeping gene, respectively. Otherwise, the steps (I) plus (II) and(III) plus (IV), respectively, may be carried out in separateexperiments.

In the methods of this invention, suitable signalling systems includefluorescence-based systems, such as wherein the self-probing ampliconfurther comprises a fluorophore/quencher pair, such as6-carboxyfluorescein/methyl red. Nevertheless, other signalling systemsmay be employed.

Preferably, amplification step(s) are carried out using the polymerasechain reaction (PCR), although the method(s) may be adapted to usealternative amplification methodology. More preferably, PCR is carriedout in ‘real time’. Accordingly, amplicon detection is preferablycarried out using real-time PCR machines, especially the iCycler(available from Bio-Rad Laboratories, UK).

The self-probing amplicon may be adapted in any suitable way known inthe art to ensure that the probe is unresponsive to amplification underthe primer amplification conditions. For example, to preventread-through to the probe component of the self-probing amplicon, theprimer component preferably further comprises an amplification blockeror stopper, such as hexethyl glycol (HEG).

Separation of the primer extension product from the target nucleic acidsequence can be undertaken by standard methods in the art. Preferably,the separation steps (II) and/or (IV) are carried out by heatdenaturation.

The target virus nucleic acid sequence is preferably one that is capableof indicating the presence of a virus that is associated with a diseaseor clinical condition in an animal, especially a mammal, more especiallyman. Particularly preferred is when the virus is human HPV. Preferably,the virus is selected from one or more of HPV types 6, 11, 16, 18, 31,33, 39, 40, 42, 43, 44, 45, 51, 52, 56, 58, 59, 66 and 68.

In such cases, the self-probing amplicon is suitably one having asequence selected from SEQ ID NOs 1 to 9, corresponding to self-probingvirus amplicons named Sc6, 11, 16, 18, 31, 33, 39, 51 and Sc56,respectively, where Scx refers to the amplicon for self-probing HPV typex (refer to Table 2 in Example 1 hereinbelow). Especially preferred iswhen the probe is the probe component of the above-noted sequences,namely a sequence selected from SEQ ID Nos [21] to [29]. (FIGS. 1 and 3refer.) 21 ATAAAGAGTACATGCGT 22 CAGATTATAAGGAATACATGC 23AGTACCTACGACATGGG 24 AGCAGTATAGCAGACATG 25 GAGTATTTAAGAGATGGTG 26CTTTATGCACACAAGAAC 27 AATATACCAGGCACGTG 28 GCAATATATTAGGCATGGG 29TCAGTACCTTAGACATGTG

Preferably, the primer is the primer component of the above-notedsequences, namely, a sequence selected from GP6+ and SEQ ID Nos: 32-40.GAAAAATAAATTGTAAATCATACTC SEQ ID NO:32 GAAAAATAAACTGTAAATCAAACTC SEQ IDNO:33 GAAAAATAAACTGTAAATCATATTC SEQ ID NO:34 GAAAAATAAACTGCAAATCATATTCSEQ ID NO:35 GAAATATAAATTGTAAATCAAATTC SEQ ID NO:36GAAAAACAAACTGTAGATCATATTC SEQ ID NO:37 GAAATATAAATTGTAAATCATACTC SEQ IDNO:38 AAAAATAAATTGCAATTCATACTC SEQ ID NO:39 GAAAAACAAATTGTAACCCATATTCSEQ ID NO:40

Suitably, the virus may be SV40, or the highly prevalent and homologousJC and BK viruses. In such a case, the self-probing amplicon ispreferably one having a sequence selected from those listed in Table 8of Example 6 hereinbelow, where Scx refers to the amplicon forself-probing SV40, JC or BK viruses (x). These sequences specificallytarget sites known to be capable of reliably detecting polyomavirusesand to be directly comparable with results obtained by other methods inthis art. Preferred forward primers are the P1 primers, as indicated inTable 8.

In the method of this invention, particularly as applied to HPV(VESPA-HPV), PCR amplification is preferably carried out using the GP5+reverse primer, as defined herein. GP5+ is a known degenerate primercapable of amplifying the 20 most common types of HPV; however, otherdegenerate primers could be used instead. Particularly suitable forcarrying out VESPA-SV40 is the use of P2 or P3 as reverse primers, asdetailed in Table 8. A preferred such method is wherein one or more ofthe amplification step(s) is/are carried out using ‘nested’ PCR

A preferred self-probing amplicon for use in viral load (per cell)determination is referred to in Table 3 in Example 2 hereinbelow as ScBG[SEQ ID No: 11]. Especially preferred is when the probe component ofsuch a self-probing amplicon is SEQ ID NO: [31]: 31 ATGGTGTCTGTTTGAG

More preferably still, the ‘viral self-probing amplicons’ include thosethat allow detection of changes in the E1, E2, E6 and/or E7 HPV genes inorder to determine integration state of the viral genome in the samplecell genome. Integration status can be measured using two differentassessment methods:

-   -   (i) Ratio of circular viral DNA to linear viral DNA; and    -   (ii) Ratio of cell cycle control viral proteins to cell        transforming proteins.

The first of these methods (i) is illustrated in FIG. 11 (as describedby Park et al in Gynecol Oncol 65(1) 121-9 (1997)). Each sample isassessed, using the method of the invention, for the quantitative amountof viral DNA in circular form. This assessment may be achieved by usingself-probing amplicons designed to contact the viral DNA before theputative viral DNA break point (in the middle of the E1 protein) and areverse primer after the putative viral break point (in the E6 or,preferably, E7 protein). This assessment reflects the quantitativeamount of viral DNA in circular form. This result may then be comparedwith total viral DNA, as determined by the method of the inventiondescribed above, to give a percentage of viral DNA in circular form.

The second of these methods (ii) relies on the fact that, uponintegration of viral DNA into the host genome, the section of viral DNAresponsible for the control of viral DNA replication and translationinto proteins is excised. This allows the proteins known to beresponsible for carcinogenesis to replicate out of control. Thus, byquantitatively measuring the ratio of the control proteins (E1 and E2)to the transforming proteins (E6 and E7), it is possible to assess thelikelihood that the sample is derived from a patient that is about toundergo malignant transformation.

Therefore, the invention further provides a method for one or more of:

(a) detection;

(b) typing;

(c) determination of viral load per cell; and/or

(d) determination of the integration state of a virus in a samplesuspected of comprising one or more target viral nucleic acidsequence(s),

which method comprises:

(IA) contacting the sample with a self-probing amplicon (‘virusself-probing amplicon’) comprising

-   -   (i) a virus primer capable of hybridising to at least one target        viral nucleic acid sequence and undergoing amplification thereof        under primer amplification conditions to form a virus primer        extension product;    -   (ii) a virus probe comprising a nucleic acid sequence        complementary to a target sequence of the virus primer extension        product and capable of hybridisation thereto, provided that the        self-probing amplicon is adapted to ensure that the virus probe        is unresponsive to amplification under the primer amplification        conditions; and    -   (iii) a member of a virus signalling system, which system is        capable of causing a detectable signal to be effected on        hybridisation of the virus probe sequence to the virus primer        extension product, whereby presence or absence of the target        viral nucleic acid sequence in the sample is indicated by the        detectable signal; and

(IB) amplifying the product of step (IA) under the primer amplificationconditions to an extent enabling the detectable signal to be effectedafter step (II);

(II) separating the virus primer extension product from the target viralnucleic acid sequence; allowing the virus probe to hybridise to thetarget sequence of the virus primer extension product; and monitoringthe signalling system,

wherein the ‘viral self-probing amplicon(s)’ is/are adapted to allowdetection, quantification or assessment of the E1, E2, E6 and/or E7 HPVgenes.

Accordingly, the present invention still further provides such ‘viralself-probing amplicons’ as are defined hereinbelow in Table 6, Example4, namely: Sc16-E1mid, Sc16-E2, Sc16-E6, Sc18-E1mid, Sc18-E2 and Sc18-E6[SEQ ID Nos: 11 to 17, respectively]. The present invention furtherprovides sequences comprising the probe part of each of these sequencesand the novel primer parts of these sequences, respectively SEQ ID NOs41 to 46 and 47 to 52. Sc16-E1mid GCAAAGAGTAATCATTA SEQ ID NO:41 probeSc16-E2 TTGTCATATAGACATATCATTTTCAT SEQ ID NO:42 probe Sc16 E6CGAATGTCTACATATCATGGC SEQ ID NO:43 probe Sc18 E1mid TCGGTGTCTCCATGTTGSEQ ID NO:44 probe Sc18-E2 TACATTGTCATGGTCTATGAT SEQ ID NO:45 probeSc18-E6 CTGGAATGCTATATCATG SEQ ID NO:47 probe Sc16-E1midCAGAATGGATACAAAGACAAACAGT SEQ ID NO:47 primer Sc16-E2CAACGTTTAAATGTGTGTCAGGA SEQ ID NO:48 primer Sc16 E6AAGTTACCACAGTTATGCACAGAGC SEQ ID NO:49 primer Sc18 E1midAGTAATGGGAGACACACCTGAGT SEQ ID NO:50 primer Sc18-E2 GCAGACACCGAAGGAAACCCSEQ ID NO:51 primer Sc18-E6 ACCCAGAAAGTTACCACAGTTAT SEQ ID NO:52 primer

Conveniently, to avoid multiple testing of a sample using many different‘virus self-probing amplicons’, is the method of the invention whereinthe target comprises more than one nucleic acid sequence from more thanone virus and/or the virus primer component exhibits some degeneracywith respect to the target, whereby the virus primer is not entirelycomplementary to each one of the nucleic acid sequences of the target.

However, for screening purposes, it may be more convenient to carry outa preliminary assay to establish first whether there is any of a virusor category of virus present in the sample and, if this assay were toprove positive, then to proceed to the specific typing and quantitationassays enabled by the method of this invention. Such a preliminary assaymay comprise any previously known method for, virus detection, includingthose mentioned hereinbefore. Preferably, however, the preliminary assaycomprises the method of the invention wherein a tailed primer is firstincorporated into the viral primer extension product. The tail comprisesa nucleic acid sequence capable of amplifying, eg under PCR conditions,the viral nucleic acid sequence of a plurality of viruses or virus, suchas HPV, types. It is believed that this is the first time the concept oftailed primers has been applied to self-probing amplicons.

Accordingly, the present invention further provides a method comprisingthe following steps:

(0)(A) contacting a target viral nucleic acid sequence from the samplewith ‘tailed primer’, which comprises:

-   -   (i) a primer region comprising a nucleic acid sequence        (‘consensus primer sequence’) complementary to a consensus        sequence of the viral nucleic acid sequence and capable of        hybridisation thereto and undergoing amplification thereof under        primer amplification conditions to form a tailed primer        extension product; and    -   (ii) a tail region comprising a unique sequence not present in        or prepared by any component of this method (‘designer’        sequence); and

(0)(B) carrying out at least two rounds of amplification under theprimer amplification conditions, whereby the ‘designer’ sequence becomesincorporated into the primer extension product; and

wherein the primer component of the ‘virus self-probing amplicon’ iscapable of binding to the ‘designer’ sequence and the probe component ofthe virus self-probing amplicon is complementary to the consensus primersequence.

Accordingly, in a preferred method of the invention in order to test forany HPV type in a single reaction, a unique self-probing amplicon targetsite is introduced into the amplification (primer extension) product. Byattaching a unique ‘designer tail’ to the prior art consensus primers(such as one capable of amplifying all 20 of the common HPV types), thisaim can be achieved. A preferred tailed primer for use in the presentinvention is shown in Table 7 of Example 5 [SEQ ID NO: 18]. Two suitabledegenerate self-probing amplicons for use in the present invention areshown in Table 7 of Example 5 [SEQ ID NOs: 19 & 20]; these sequencesalso comprise preferred primer and probe components. (FIGS. 6 and 7refer.)

Accordingly, the present invention further provides the novel tail part[SEQ ID NO: 10] of these sequences; the probe part of these sequencesbeing designed to bind to the known GP6+ sequence. ATGTGGAAACATGCATGGGP6+ tail comprised SEQ ID NO:10 in [SEQ ID NO:18]

Especially preferred is when the method of the invention is incorporatedinto a screening programme and the self-probing amplicons of thisinvention are for use therein.

Accordingly, the invention still further provides:

(a) A screening method for screening an individual suspected of a viralinfection, which screening method comprises:

-   -   (i) obtaining a sample of a nucleic acid sequence from the        individual; and    -   (ii) carrying out, on the sample, a method according to the        invention as described above, whereby presence of the detectable        signal from the virus signalling system indicates presence of        the viral infection and absence of the detectable signal        indicates absence of the viral infection;

(b) A screening method according to (a), which screening method furtherindicates the presence or absence of specific viral type(s);

(c) A screening method according to (a) or (b), which screening methodfurther indicates viral load per cell, when virus is present (being zerowhen virus is absent);

(d) A screening method according to any of (a) to (c), which screeningmethod further indicates integration status of the virus, when present;

(e) A screening method according to any of (a) to (d), which screeningmethod is adapted for screening for cervical cancer, recurrentrespiratory papillomatosis or any other condition associated with thepresence in the individual of a human papillomavirus (HPV); and

(f) A screening method according to any of (a) to (e), which screeningmethod is adapted for screening for one or more of: mesotheliomas,including cancers of the chest and lung; osteosarcomas; pituitary,thyroid, brain and neurological tumours (eg glioblastomas, astrocytomas,ependymomas and papillomas of the choroids plexus); and other conditionsassociates with SV40, JK and/or BK virus(es).

The methods of the invention, although preferably carried out on DNA,can be adapted by methods known to those skilled in the art to becarried out on RNA. In this case, any commercially available reversetranscriptase could be employed first to transcribe the RNA to DNA, andthen the method carried out as described above.

The present invention also provides a diagnostic kit for use in a methodof the invention, which kit comprises one or more of the virusself-probing amplicons, housekeeping self-probing amplicons or tailedprimers of the invention. Particularly preferred is a kit suitable foruse in a method according to the invention for determining viral loadper cell, and accordingly such comprises at least two, and suitably fouror more, self-probing amplicons, including at least one housekeepingself-probing amplicon and, optionally, a tailed primer. Also preferredis a kit comprising at least one self-probing amplicon for targeting atleast one of the E1, E2, E6 and E7 HPV genes.

Many of these self-probing amplicons are themselves new and inventive.Accordingly, the invention further provides:

(a) a novel self-probing amplicon as described herein, including:

-   -   (i) SEQ IDs NOs: 1-9 (Sc 6, 11, 16, 18, 31, 33, 39, 51 & 56)    -   (ii) SEQ ID NO: 11 (ScBG)    -   (iii) SEQ ID NOs: 12-17 (Sc16-E1mid, Sc16-E2, Sc16 E6, Sc18 E1        mid, Sc18-E2, Sc18-E6)    -   (iv) SEQ ID NOs: 18-20 (tailed primers)    -   (v) Sequences specified in Table 8 hereinbelow (self-probing        amplicons for use in SV40, JC or BK determination)

(b) a novel probe component of (a), including:

-   -   (i) SEQ IDs NOs: 21-29 (probe components of (Sc 6, 11, 16, 18,        31, 33, 39, 51 & 56)    -   (ii) SEQ ID NO: 31 (probe component of ScBG)

(iii) SEQ IDS NOs: 41-46 Sc16-E1mid GCAAAGAGTAATCATTA SEQ ID NO:41 probeSc16-E2 TTGTCATATAGACATATCATTTTCAT SEQ ID NO:42 probe Sc16 E6CGAATGTCTACATATCATGGC SEQ ID NO:43 probe Sc18 E1mid TCGGTGTCTCCATGTTGSEQ ID NO:44 probe Sc18-E2 TACATTGTCATGGTCTATGAT SEQ ID NO:45 probeSc18-E6 CTGGAATGCTATATCATG SEQ ID NO:46 probe

(iv) SEQ IDS NOs: 59 and 60, being the probe component of SEQ ID NOs:19-20 (tailed primers) GAAGAATATGATTTACA SEQ ID NO:59 GAGGAATATGATTTACASEQ ID NO:60

(v) SEQ ID Nos: 53 to 55, being the probe component of the sequenceslisted in Table 8 (SV40, BK and JC self-probing amplicons);ACCCCAAGGACTTTCCT SEQ ID NO:53 CCTATGGAACAGATGAATG SEQ ID NO:54ACCCTAAAGACTTTCCC SEQ ID NO:55

(c) a novel primer component of (a), including:

(i) SEQ IDS NOs: 3240 (primer components of Sc 6, 11, 18, 31, 33, 39, 51& 56), respectively: GAAAAATAAATTGTAAATCATACTC SEQ ID NO:32GAAAAATAAACTGTAAATCAAACTC SEQ ID NO:33 GAAAAATAAACTGTAAATCATATTC SEQ IDNO:34 GAAAAATAAACTGCAAATCATATTC SEQ ID NO:35 GAAATATAAATTGTAAATCAAATTCSEQ ID NO:36 GAAAAACAAACTGTAGATCATATTC SEQ ID NO:37GAAATATAAATTGTAAATCATACTC SEQ ID NO:38 AAAAATAAATTGCAATTCATACTC SEQ IDNO:39 GAAAAACAAATTGTAACCCATATTC SEQ ID NO:40

(iii) SEQ IDS Nos: 47-52, in particular, the E1 mid primer sequences[SEQ ID Nos: 47 and 50]: Sc16-E1mid CAGAATGGATACAAAGACAAACAGT SEQ IDNO:47 primer Sc16-E2 CAACGTTTAAATGTGTGTCAGGA- SEQ ID NO:48 primer Sc16E6 AAGTTACCACAGTTATGCACAGAGC SEQ ID NO:49 primer Sc18 E1midAGTAATGGGAGACACACCTGAGT SEQ ID NO:50 primer Sc18-E2 GCAGACACCGAAGGAAACCCSEQ ID NO:51 primer Sc18-E6 ACCCAGAAAGTTACCACAGTTAT SEQ ID NO:52 primer

(iv) SEQ IDS NO: 61 being the primer component of SEQ ID NOs: 19-20(tailed primers) SEQ ID NO: 61 GTGGAAACATGCATGGCGAC

(v) SEQ ID Nos: 56 to 58, being the primer component of the sequenceslisted in Table 8 (SV40, BK and JC self-probing amplicons); SEQ ID NO:56 AGCATGACTCAAAAAACTTAGCAATTCT SEQ ID NO: 57TTCTCATTAAATGTATTCCACCAGGATT SEQ ID NO: 58 AGCTTGACTAAGAAAGTGGTGTAGATCA

(d) [SEQ ID NO: 10] ATGTGGAAACATGCATGG tail sequence of GP6+ tailedprimer

Furthermore, the invention provides the use of such self-probingamplicons, including the probe and primer components thereof, in amethod or in the preparation of a kit as hereinbefore described.

The present invention will now be illustrated by the following Examples.

EXAMPLE 1 Viral Detection Using VESPA

General Materials and Methods

Cell Lines

The HeLa, Caski and SiHa cell lines were provided by Dr Steve Man,University of Wales College of Medicine, Cardiff, UK, and are availablefrom the ATCC (American Type Culture Collection, http://www.atcc.org/)

Clinical Samples

Patients were either recruited during routine colposcopy clinics atLlandough Hospital, Cardiff, UK, or as part of an MRC field study inWest Africa. Informed consent was obtained from all subjects who wereasked for permission to assess their smear for HPV, but the methodologyremained confidential. Cervical samples were collected using conicalcytobrushes, and transported in 0.5 ml of Digene transport medium (fromSilver Spring, Md., USA). Samples taken in the UK were stored at 4° C.for up to 24 hours before processing. Samples collected in the Gambiawere stored, frozen in liquid nitrogen, and processed within one month.Biopsies were collected, with informed consent, from patients undergoingtreatment for Recurrent Respiratory Papillomatosis and cervical cancerat The University Hospital of Wales, Cardiff, UK and Llandough Hospital,Penarth, UK.

DNA Purification

DNA was purified from cell lines by re-suspension of cells in 640 μl ofNuclear Lysis Buffer (10 mM Tris HCl, 0.4M NaCl, 2 mM ethylenediaminetetra-acetate pH 8.0, 10% sodium dodecyl sulphate), 100 μl of 6M NaCland 740 μl of chloroform. The solution was thoroughly mixed, centrifugedand the top phase extracted. DNA was precipitated by the addition of 1ml 95% ethanol and pelleted by centrifugation. The pellet was washedtwice with 70% ethanol, dried in a rotary evaporator and re-suspended in500 μl of de-ionised water. DNA was purified from cervical brush samplesby a simple modification (squeezing the cytobrushes on the side of thetube, and freezing times were increased to 24 hours from 2 hours) of thefreeze-thaw method of described by Jacobs et al in Jacobs et al J ClinMicrobiol 35 791-5 (1997). Epithelial cells obtained from the cytobrushsamples were pelleted by centrifugation and re-suspended in 1 ml 10 mMTris pH 7.4, and frozen at −70° C. for 24 hours. A 100 μl aliquot wasthawed, boiled for 10 minutes; chilled on ice; spun at 13,000 rpm in amicrofuge for 3 minutes; and supernatant decanted and stored. DNA wasextracted from biopsy material using a modification of the abovetechnique, in which samples were incubated in 1 ml 10 mM Tris HCl pH7.4containing 10 mg/ml proteinase K (from Sigma, UK) for 1 hour at 56° C.before boiling.

PCR EIA—Comparative Method

PCR-EIA was performed as described by Jacobs et al (q.v.).

VESPA Method

Primer Design

Table 2 shows the sequences of the ten self-probing amplicon primersused in this study. All primers were synthesised by Oswel ResearchProducts, Southampton SO16 7PX, UK.

All self-probing amplicon primers were purified by double HPLC. Standardprimers were purified by gel filtration. Primers were designed to detectthe HPV L1 gene. Primer locations varied between types but werepositioned approximately between 6600 and 6750 bp.

The forward primer sequence of each self-probing amplicon istype-specific, and is located at the same sequence position as that ofthe GP6⁺ primer described by Jacobs et al (q.v.). Self-probing ampliconprobe sequences were designed by aligning the L1 open reading frames(ORF) of twenty common HPV types (HPV-6, 11, 16, 18, 31, 33, 39, 40, 42,43, 44, 45, 51, 52, 56, 58, 59, 66 and 68) (http://hpv-web.1an1.gov).The areas of greatest sequence variation adjacent to the GP6⁺ primerbinding site of Jacobs et al (q.v.) were selected as the probe targetbinding site. The probe sequence of these primers was checked against 70common papillomavirus sequences and no significant homology was found.The SC16 primer component comprises the known GP6+ sequence. The reverseprimer target sequence is the GP5+ sequence of Jacobs et al (q.v.).TABLE 2 Sequences of VESPA Primers. Typing SEQ ID Primers NO: SequenceHPV-6 Sc6 1CCGCGGATAAAGAGTACATGCGTCCGCGG-MR-HEG-GAAAAATAAATTGTAAATCATACTC HPV-11Sc11 2 CCCGCGGCAGATTATAAGGAATACATGCCCGCGGG-MR-HEG-GAAAAATAAACTGTAAATCAAACTC HPV-16 Sc16 3CCGCGGAGTACCTACGACATGGGCCGCGG-MR-HEG-GAAAAATAAACTGTAAATCATATTC HPV-18Sc18 4 CCGCGGCAGCAGTATAGCAGACATGGCCGCGG-MR-HEG-GAAAAATAAACTGCAAATCATATTC HPV-31 Sc31 5CCCGCGGGAGTATTTAAGACATGGTGCCGCGGG-MR-HEG- GAAATATAAATTGTAAATCAAATTCHPV-33 Sc33 6CCGCGGCTTTATGCACACAAGAACCCGCGG-MR-HEG-GAAAAACAAACTGTAGATCATATTC HPV-39Sc39 7 CCCGCGGAATATACCAGGCACGTGCCGCGGG-MR-HEG-GAAATATAAATTGTAAATCATACTCHPV-51 Sc51 8CCCGCGGGCAATATATTAGGCATGGGCCGCGGG-MR-HEG-AAAAATAAATTGCAATTCATACTC HPV-56Sc56 9 CCGCGGTCAGTACCTTAGACATGTGCCGCGG-MR-HEG-GAAAAACAAATTGTAACCCATATTCReverse 5′-TTTGTTACTGTGGTAGATAC Primer-GP5+HPVn = human papillomavirus n; Sc = self-probing amplicon; MR =Fluorescence quencher methyl red; HEG = PCR blocker = hexethyl glycol.Sequences in bold typeface are novel probe sequences.Primer Validation—VESPA v Reference Cell Lines

The HPV 16 and 18 primers were tested for specificity using referencecell lines with integrated HPV DNA. The Caski cell line contains 60-600copies of the HPV-16 ORF per cell, and the HeLa cell line contains 10-50copies of the HPV-18 L1 ORF.

FIG. 1 a shows the results of PCR reactions using the Sc16 self-probingamplicon primer (designed for detection of HPV-16 DNA), and DNAextracted from the HPV-16 positive Caski cell line, DNA from the HPV-18positive HeLa cell line and a negative control (no DNA). A significantincrease in fluorescence was only detected with the HPV-16 containingCaski DNA.

FIG. 1 b shows a similar experiment using the Sc18 self-probing ampliconprimer in place of Sc16. Here, significant fluorescence was onlydetected with the HPV-18 positive HeLa cell line. The primers were thenused to detect HPV-16 and -18 in clinical samples previously typed usingPCR-EIA (FIG. 2 c and FIG. 2 d—see below).

Detection of HPV by VESPA

PCR amplification of 1 μl aliquots of DNA solution were performed using0.5 μM self-probing amplicon primer and 0.5 μM GP5⁺ reverse primer asdescribed by Jacobs et al (q.v.) in a total reaction volume of 10 μl.Reactions were performed using a Light Cycler (available from Bio/Gene,Kimbolton, Cambs, PE18 0NJ or Roche Diagnostics Ltd, Bell Lane, Lewes,East Sussex BN7 1LG) and run for 100 cycles under the following cyclingparameters: 96° C.-1s, 40° C.-5s, 72° C.-1s. Reaction conditions were asfollows: 200 μM dNTP's, 4 mM MgCl₂, 50 mM Tris HCl pH 8.9, 10 mMammonium sulphate, 0.1% Tween™ 20, bovine serum albumin 250 ng/μl, and0.5 U/μl Taq polymerase (from Advanced Biotechnologies, Epsom, Surrey,UK). Fluorescence was detected in channel one (530 nm) at 40° C.

PCR control reactions were carried out as above but included 1 μl SYBRGold (from Bio/Gene, Kimbolton, Cambs, PE18 0NJ). Self-probing ampliconcontrol reaction (negative controls) contained 1 μl H₂O in place of DNA.

The VESPA technique was then extended to enable detection of HPVtypes-6, -11, -31, -33, -39, -51 and -56. As there are nocommercially-available cell lines containing these HPV types, primerspecificity was validated using clinical samples previously tested usingPCR-EIA as shown below in Table 5, with reference to Example 3. FIG.2(a-h) shows positive results from typing reactions for HPV-6, -11, -16,-18,-31, -33, -39, and -51, respectively.

EXAMPLE 2 Viral Load Determination Using VESPA

A theoretical advantage of self-probing amplicon PCR is its ability todetermine viral load. Shown, in FIG. 3, are the results of Sc16 typingreactions performed using a dilution series of the SiHa cell line (oneto two copies of HPV-16 per cell). The dilution series from 50,000 to500 HPV copies per cell were clearly distinguishable, and the signal forHPV-16 remained positive in the sample containing a single copy ofHPV-16 DNA.

In order to explore the possibility of establishing a technique capableof determining viral load per cell, a primer was designed to detect(human) beta-globin DNA (ScBG, SEQ ID NO: 11 in Table 3). TABLE 3 HPV-16Viral Load - Sequences of VESPA Primers. Beta- Globin Detection SEQ IDPrimer NO: Sequence ScBG 11 FAM-CCGCGGATGGTGTCTGTTTGAGCCGCGG-MR-HEG-ACACAACTGTGTTCACTAGC Reverse 5′-GAAACCCAAGAGTCTTCTCT-3′ PrimerSc = self-probing amplicon; FAM = Fluorophore = 6-carboxyfluorescein. MR= Fluorescence quencher = Methyl Red; HEG = PCR blocker = hexethylglycol.Sequences in bold typeface are novel probe sequences

FIG. 4 shows the results of an experiment conducted using a self-probingamplicon designed to detect the human beta-globin gene over the samedilution series of SiHa cells used above for HPV-16. Once again, thesignal remained positive down to a single cell and is quantitative atand above 50 copies per cell. FIG. 5 shows a plot of the ratio of thefluorescence produced by Sc16 and ScBG against the logarithm of theviral copy number per cell of target DNA. A range of copy numbers percell may be calculated from a cell line containing a fixed copy number,by calculating the ratio of two different dilutions. For example, theSc16 F_(max) value for 5000 cells can be divided by that for the ScBGF_(max) for 100 cells to obtain a value for a notional cell linecontaining 50 copies per cell (5000/100).

Disease grade was determined by cytology. VL ratio (Viral Load) wasmeasured in RSU Relative Self-probing amplicon Units) by dividing Sc16F_(max) by ScBG F_(max). Viral copies per cell were estimated using thestandard curve shown in FIG. 5 TABLE 4 Viral Load Data HPV- Disease VLratio Av. Viral Copies Sample Type Grade (RSU) per cell 1 2885C 16Normal −0.3508 Undetectable 2 2503F 16 Normal −0.1204 Undetectable 30155H 16 Normal −0.1007 Undetectable 4 LD20 6,16,39 CIN2 −0.0242Undetectable 5 0119D 16 Normal −0.0166 Undetectable 6 SJ 16 Ca vagina0.0095 0.042 7 LD49 16 CIN2-3 0.0331 0.047 8 AD 16 CIN2-3 0.0550 0.052 9LD19 16 CIN2 0.0697 0.056 10 LD15 16 CIN3 0.0750 0.057 11 LD25 16,31CIN3 0.0786 0.058 12 LD24 16 CaCx 0.1641 0.088 13 LD50 mm 16 CIN3 0.49450.441 14 2503F 16 CIN2-3 0.5965 0.724 15 LD45 16 CIN2-3 0.7601 1.603 160093A 16 CIN2-3 0.8749 2.805

Shown in Table 4 are the results of applying this viral loaddetermination technique to 16 clinical samples previously found (byJacobs et al, q.v.) to be HPV-16-positive using PCR-EIA. The moststriking finding from these experiments is that the four cervical smearswith normal cytology have low viral loads using VESPA, and the onlyother sample with a low viral load, but significant neoplasia, isco-infected with HPV-6 and HPV-39. The preliminary viral load data shownin Table 4 suggest that the presence of cervical neoplasia mightcorrelate with viral load.

EXAMPLE 3 Comparison of VESPA With PCR-EIA

In order to test the suitability of VESPA for HPV typing from cervicalsmears in the clinical setting, the self-probing amplicon primers wereused to test 108 samples previously HPV typed (Jacobs et al q.v.), usingPCR-EIA. In order directly to compare the two techniques, DNA extractionwas performed using the freeze-thaw method described by Jacobs et al,q.v. This technique is sub-optimal for PCR amplification usingself-probing amplicon primers. The less efficient freeze-thaw DNAextraction method was used in this study, since this method isrecommended for PCR-EIA and we wished to compare PCR-EIA and VESPA usingidentical DNA samples. The results of these experiments are shown inTable 5.

Selected positive sample types have subsequently been confirmed bydirect sequencing (data not shown). The overall concordance betweenVESPA and PCR-EIA is 94%, with a kappa value of 0.89 indicating goodagreement. Indeed, a similar concordance figure was achieved whenPCR-EIA was performed on identical samples by different referencelaboratories described by Jacobs et al q.v. Of 108 samples, VESPA failedto detect five incidences of HPV-16 and one of HPV-18. There were nofalse positives.

There are several explanations for the small number of remainingdiscrepancies observed between VESPA and PCR-EIA. First, the resultsproduced by PCR-EIA might be false positives. There is evidence from thestudy comparing PCR-EIA in several different laboratories that it isprone to the occasional false positive. The fact that four of the sixdiscrepant samples have normal cytology supports this supposition. Ofthe remaining two, one has low-grade lesions and for the other we haveno clinical data. Secondly, the samples may be positive for HPV butcontain an intra-typic HPV variant containing polymorphism within theprobe-binding site. Self-probing amplicon probes discriminate betweensequences on the basis of a single base change, whereas PCR-EIA probesare more tolerant of sequence variation. In this regard, it may berelevant that 5/6 discordant samples were obtained from West Africa,where there is likely to be more variation within the probe bindingsite. TABLE 5 Comparison of PCR-EIA and VESPA KAPPA Sample Type HPVType(s) PCR-EIA VESPA VALUES Cell Line 16 2 2 n/a (n = 3)  18 1 1 Biopsy6 1 1 n/a (n = 3)  11 2 2 UK Cervical Samples 16 8 7 33 2 2 (n = 54)16,31 1 1 16,39 1 1 0.95 6,16,39 1 1 33,51 1 1 neg 40 41 AfricanCervical 11 4 4 Samples (n = 48) 16 7 3 16,31 1 1 18 6 5 31 2 2 0.79 332 2 39 2 2 51 3 3 56 3 3 neg 18 23 Overall 0.89 (n = 108)

k-values were calculated as described by Armitage et al in StatisticalMethods in Medical Research, Third Edition (Oxford; Blackwell ScientificPublications)

EXAMPLE 4 VESPA to Detect Integration Status—Calculation of E6/E7:E1/E2

Self-probing amplicon sequences designed for the assessment of HPVintegration state are shown in Table 6. They are used in accordance withthe method described in the Examples above. Integration status ismeasured as described hereinabove. TABLE 6 Integration Primers SEQ IDPrimer NO: Sequence Sc16- 12 5′-FAMCCGCGGGCAAAGAGTAATCATTACCGCGG-MRE1mid HEG-CAGAATGGATACAAAGACAAACAGT-3′ RP16 E15′-GCGCATGTGTTTCCAATAGTCTA-3′ Sc16-E2 135′-FAMCCCGCGGTTGTCATATAGACATATCATTTT- CATCCGCGGG-MR-HEG-CAACGTTTAAATGTGTGTCAGGA-3′ RP16 E2 5′-AGACACACAAAAGCACACAAAAGC-3′ Sc16E6 14 5′-ROXCCGCFAMTGGCGAATGTCTACATATCATGGC- CCAGCGG-MR-HEG-AAGTTACCACAGTTATGCACAGAGC-3′ RP16 E7 5′-GAGAACAGATGGGGCACACAAT-3′ Sc1815 5′-FAMCCCGCGGTCGGTGTCTCCATGTTGCCGCGGG- E1midMR-HEG-AGTAATGGGAGACACACCTGAGT RP18 E1 5′-AGTGGTCTATGATTTTGTCCTGCA-3′Sc18-E2 16 5′-FAMCCGCGGTACATTGTCATGGTCTATGAT-CCGCGG-MR-REG-GCAGACACCGAAGGAAACCC-3′ RP18-E25′-GCATACACAAAAGCAAAATAAAAAA-3′ Sc18-E6 175′ROXCCGCFAMTGGCTGGAATGCTATATCATGCCA-GCGG-MR-HEG-ACCCAGAAAGTTACCACAGTTAT-3′ RP18-E7 5′CCGTCTGTACCTTCTGGATC-3′Key:Sc = self-probing amplicon; RP = reverse primerROX = ROX; FAM = Fluorophore = 6-carboxyfluoresceinMR = Fluorescence quencher = Methyl RedHEG PCR blocker = hexethyl glycolBold typeface indicates probe and primer sequences, respectively.

EXAMPLE 5 VESPA Using Tailed Primers for Degenerate HPV Detection

A suitable strategy comprises the following steps, illustrated by FIG.6:

1. A primer containing a ‘designer tail’ is used to amplify viral DNA(if present). A standard reverse primer is also included.

2. Amplification proceeds as in a standard reaction.

3. After the first round of amplification, the ‘designer’ sequence hasno complementary sequence.

4. After the second round of amplification, the ‘designer’ sequence isincorporated and is amplified in each round.

5. In a separate reaction, this amplification can then be detected usinga self-probing amplicon comprising a primer targeting the ‘designer’sequence and a probe for detecting the consensus primer. It may benecessary to use two self-probing amplicons with a slight overhang intothe viral DNA to avoid unacceptable background fluorescence caused byprimer dimer.

Using a method analogous to that described in previous Examples, thefollowing tailed primers were used to detect degenerate HPV (Table 7):TABLE 7 Tailed Primers SEQ ID Primer NO: Sequence Tailed 18ATGTGGAAACATGCATGGCGAGATGAAAAATAAACTG GP6+ TAAATCATATCT ScDGi 19FAM-CCGCGGGAAGAATATGATTTACACCGCGG-MR- HEG-GTGGAAACATGCATGGCGAC ScDGii 20FAM-CCGCGGGAGGAATATGATTTACACCGCGG-MR- HEG-GTGGAAACATGCATGGCGACKey:Sc = self-probing amplicon; FAM = Fluorophore 6-carboxyfluoresceinMR = Fluorescence quencher = Methyl Red; BEG = PCR blocker = hexethylglycolSequence in bold typeface is novel tail region. Both primer and proberegions are also novel.

FIG. 7 demonstrates the ability of the degenerate self-probing ampliconmix to detect HPV types-6, -16 and -18. Comparisons may be performedusing samples pre-amplified under conditions described by Jacobs et al(qv). Interestingly, two of these samples were barely visible afteragarose gel electrophoresis with ethidium bromide staining, suggestingthat the VESPA approach produces the expected improvement insensitivity.

EXAMPLE 6 VESPA Applied to SV40

DNA Extraction

Ten sequential 5 μM sections of paraffin-embedded mesothelioma biopsieswere cut, taking precautions to avoid block-to-block contamination. Thesections were placed in microfuge tubes and treated with xylene for 10mins in order to remove paraffin, then washed twice with ethanol anddried. 400 μl of digestion buffer containing 200 μg/ml of proteinase Kwere added and the specimens digested overnight at 55° C. Proteinase Kwas then inactivated by heating to 95° C for 10 mins. The digests werefurther processed for DNA extraction using phenol/chloroform/isoamylalcohol. DNA was precipitated with ethanol, centrifuged and air dried.Further de-salting and concentration of DNA was performed usingCentricon 30 filters (Millipore, Bedford, Mass., USA). The pure DNA wasquantified spectrophotometrically. All samples tested positive forbeta-globin amplification (Muggleton-Harris et al. Hum Reprod 10 183-92(1995)), indicating PCR viability.

Primer Design

Primers were designed by aligning the sequence of each virus.(http://www.ncbi.nlm.nih.gov/PubMed/). SV40 Accession Number J02400; JCAccession Number J02226/7; BK Accession Number NC 001538. For the firstround amplification, several primers were designed, throughout the largeT antigen gene (see Table 1), using established, historical primersites. For the first round amplification step, because of thesubstantial degradation of the DNA found in most samples, it waspreferred to use primers capable of targeting short DNA fragments inwhich the target sites were close together. Self-probing amplicon primerand probe binding sites were designed to target areas of least homologybetween these primer sites.

Table 8: Sequences of SV40-VESPA Primers

All self-probing amplicon primers were purified by double hplc. Standardprimers were purified by gel filtration. Primers were designed to detectthe large T antigen.

Key:

FAM=Fluorophore=6-carboxyfluorescein

MR=Fluorescence quencher=Methyl Red

HEG=PCR blocker=hexethyl glycol

The approximate position of primers and probes is also shown. P1indicates a forward primer; P2 and P3 are reverse primers. TABLE 8 NameLocation Sequences of SV40-VESPA Primers & Probes ScsSV40 4361- a g ca tg a c t c a a a a a a c t t a g c a a t t c t primer 4388 ScJC primer4361- t t ct c a t t a a a t g t a t t c c a c c a g g a t t 4388 ScBKprimer 4347- a g ct t g a c t a a g a a a c t g g t g t a g a t c a 4374ScSV40 4390- a c ccc a a g g a c t t t c c t probe 4406 Sc JC probe4390- c c ta t g g a a c a g a t g a a t g 4406 ScBK probe 4376- a cc ct a a a g a c t t t c c c 4392 SV40 P1 4318- t t tt c c t t t g t g g tg t a a a t 4337 SV40 P2 4921- a c tt t g g a g g c t t c t g g g a t gc a a c 4945 SV40 P3- 4371- a g at t c c a a c c t a t g g a a c t g a t4398 SV40 P4 4453- g a gg c t a c t g c t g a c t c t c a a c a t 4476JC P1 4318- t t tc a t c a t c a c t g g c a a a c 4337 JC P2 4921- c atgg g g g a a c a t t c c t g t c a t g a g 4945 JC P3 4371- g t gt g ac t t a a c c c a a g a a g c t c 4398 JC P4 4453- t t t gc a g g g c at t t t g t t t t t t a c 4476 BK P1 4550- t t tt c t t t a g t a g t at a c a c 4574 BK P2 4453- t g ga a c c t g g a g t a g c t c a g a g gt t 4476 BK P3 4908- c c ta t g g a a c a g a a g a g t g g g a g 4932BK P4 4439- g a a gc a a c a g c a g a t t c t c a a c a c 4463Assay Conditions

All amplifications were carried out using good laboratory practise,filter tips and in a laboratory not previously exposed topolyomaviruses.

First Round Amplification (Pre-amplification).

Each reaction was set up three times with appropriate primer pairs inorder to test for SV40, JC and BK.

5 μl of each sample per reaction was pre-amplified under the followingconditions; 0.5 μM P1 and P3 (see Table 8), 2.5 mM MgCl₂, 200 μM dNTPs,1.25 U of Taq polymerase (Advanced Biotechnologies, Epsom, Surrey, UK)and 5 μl of 10× reaction buffer (Perkin-Elmer Gold) in a total reactionvolume of 50 lμ.

Each sample was also SV40-typed under the conditions described above andusing the P1-P2 primer pair (Table 8).

Second Round Amplification (Amplicon Detection).

Assay set-up was performed in a laboratory physically separated (100 m)from that used for the first round amplification.

Amplicon detection was performed in two different real-time PCRmachines:

(i) LightCycler (Roche Diagnostics Ltd, Bell Lane, Lewes, East SussexBN7 1LG) and

(ii) iCycler (Bio-Rad Laboratories Ltd., Bio-Rad House, Maylands Avenue,Hemel Hempstead, Herts. HP2 7TD); and under slightly differentconditions.

PCR amplification of 1 μl aliquots of DNA (2 μl iCycler) solution wereperformed using 0.5 μM Scorpion primer (see Table 1) and 0.5 μM reverseprimer in a total reaction volume of 10 μl (20 μl iCycler). Reactionconditions were as follows: 200 μM dNTPs, 4 mM MgCl₂, 50 mM Tris HCl pH8.9, 10 mM ammonium sulphate, 0.1% Tween 20, bovine serum albumin 250ng/μl, and 0.5 U/μl Taq polymerase (Advanced Biotechnologies, Epsom,Surrey, UK). Fluorescence was detected in channel one/530 nm (FilterSet4iCycler) and at 40° C. (45° C. iCycler). Self-probing amplicon controlreaction (negative controls) contained H₂O in place of DNA. All primerswere synthesised by Oswel Research Products, Southampton SO16 7PX, UK.

Cycling Parameters

Pre-Amplification

94° C. for 180 s followed by 30 cycles of 94° C.-60 s, 52° C.-60 s and72° C.-60 s and finally 72° C.-300 s.

Amplicon Detection

(i) LightCycler. 100 cycles of 96° C.-1 s, 40° C.-5 s, 72° C.-1 s.

(ii) iCycler. 95° C. for 180 s followed by 40 cycles of 95° C.-30 s, 45°C.-30 s, 72° C.-15 s.

Results

Shown in FIGS. 10 a-10 c are the results of challenging the self-probingamplicon designed for the detection of SV40 (ScSV40), that designed forthe detection of JC virus (ScJC) and that designed for the detection ofBK virus (ScBK) with pre-amplified cell line DNA, mesothelioma-derivedDNA and water in a LightCycler real time fluorimeter. A significantincrease in fluorescence is observed only when a specific self-probingamplicon encounters a cognate virus. Background levels of fluorescencedue to non-cognate virus remains low in all cases bar ScBK whenchallenged with JC virus.

FIGS. 10 d-10 f show equivalent reaction profiles when analysed using aniCycler real time fluorimeter. FIGS. 10 e and 10 f do not show reactionprofiles produced by samples. Fluorescence is strong when a specificself-probing amplicon is presented with cognate virus and weak withnon-cognate virus. Background fluorescence due to non-cognate virus is,however, increased in this series of experiments.

Mesothelioma viral typing results, using all three self-probingamplicon, two ScSV40 target amplicons and two different modes ofamplicon analysis, are shown in Table 9.

Table 9: Analysis of Mesothelioma-Derived DNA Using SV40-VESPA

Positives were assigned to those reactions producing at least threetimes background fluorescence (ie 3× that produced by a blank reactioncontaining only water or background fluorescence due to non-cognatevirus, whichever is higher). TABLE 9 Analysis of Mesothelioma-derivedDNA using SV40-VESPA Positives were assigned to those reactionsproducing at least three times background fluorescence (ie 3 x thatproduced by a blank reaction containing only water or backgroundfluorescence due to non-cognate virus, whichever is higher). ScorpionType SV4D JC BK Amplicon analysed P1-P2 P1-P3 P1-P3 P1-P3 Mode ofAnalysis Rodu et al LightCycler LightCycler iCycler LightCyclerLightCycler Sample Name Positive Control pos pos pos pos pos Blnk negneg neg neg neg 1 neg pos pos neg neg 2 neg neg neg neg neg 3 neg insufinsuf insuf insuf 4 neg insuf insuf insuf insuf 5 neg pos pos neg neg 6pos pos pos neg neg 7 neg pos pos neg pos 8 neg pos pos neg neg 9 negpos pos neg neg 10 neg neg neg neg neg 11 neg pos pos neg neg 12 neg negneg neg neg 13 neg pos pos pos neg 14 neg neg neg neg neg 15 neg pos posneg neg 16 neg neg neg neg neg 2059 neg neg neg neg neg W335 neg pos posneg neg GFPM neg pos pos pos neg S84 neg pos pos pos neg 10684 neg pospos neg neg 495-2905 neg pos pos neg neg 6813 neg pos pos neg neg36/96-2 neg neg neg neg neg 2341 neg neg neg neg neg 594 neg neg neg negneg 286 700 6 neg neg neg neg neg 286 700 4 neg pos pos neg neg F96 3-5neg neg neg pos neg 951-4-6 neg pos pos neg neg P9800162F neg pos posneg neg 184/92H neg pos pos neg neg P96/3473 neg pos pos neg neg 634/94Aneg pos pos pos neg 103/95 neg pos pos pos neg BP 9459 neg pos pos negneg Positive Control pos pos pos pos pos Blnk neg neg neg neg negPositive Control pos pos pos pos pos Blnk neg neg neg pos neg PM149/9322 neg pos pos neg neg 6656/98B neg pos pos neg neg 96N 129B negpos pos pos neg 10 330/94 R2 neg pos pos neg neg 95-67-P neg neg neg negneg D94-7M neg pos pos neg neg S84/93 neg pos pos pos neg 634/94A negneg neg neg neg 95/10684I neg neg neg neg neg 495-2905 neg neg neg negneg P94-212 neg pos pos neg neg M93-268-1 neg neg neg neg neg F98-29-4neg pos pos neg neg 5326-91 neg neg neg neg neg F237-91 neg neg pos negneg 119M-94 neg neg pos neg neg 3226-91 neg neg pos neg neg A-352-91 negneg pos neg neg 630-91-5 neg neg neg neg neg 3009-91 neg neg pos neg negA17886/95 neg neg pos neg neg 95N26 neg neg pos neg neg 9839/97 neg pospos neg neg 537-94-7 neg pos pos pos neg 96/746A neg pos pos pos neg14839/94 8 neg pos pos neg neg 128/93 neg pos pos pos neg A3087-91 negpos pos neg neg 7327/94 neg pos pos pos neg 93/3012 neg pos pos pos negTP 247/94 neg neg neg pos neg 97-0818D neg pos pos neg neg 10947/94 negpos pos neg neg 93-300 neg pos pos neg neg M93606A neg neg neg neg negP93 6249/4 neg pos pos neg neg B476A93 neg neg pos neg neg P2 54/92 negneg neg neg neg E9254/92/10 neg neg neg neg neg A414/1/92 neg pos posneg neg Positive Control pos pos pos pos pos Blnk neg neg neg neg negPositive 1 44 52 14 1 Negative 75 30 22 60 73 % incidence 1.30% 59% 70%19% 1.40%

These are the typing results of 76 archival mesothelioma DNA samplesderived from paraffin embedded blocks. There are several observations ofnote. First, analysis targeting a 650 bp amplicon between P1 and P2 (seeTable 8) produces a single positive, whereas analysis targeting a 230 bpamplicon between P1 and P3 produces 44 positives. This observation ismost likely due to the fact that often only short sections of DNA may berecovered from paraffin-embedded blocks but may also reflect the factthat SV40 has become integrated into the host genome in these sampleswith concomitant DNA deletion.

Secondly, comparing SV40-VESPA and established methodology reveals anincrease in the number of positives observed suggesting an increase insensitivity. This comparison is dependent on the mode of fluorescencemeasurement. SV40 incidences of 1.3% were found when analysing ampliconP1-P2 (650 bp); 59% when analysing amplicon P1-P3 (230 bp) using aLightCycler; and 70% when analysing amplicon P1-P3 using an iCycler.Thus, data produced using an iCycler reports nearly 20% more positivesthan that reported by a LightCycler. Background fluorescence is,however, increased using the iCycler. This increase in the observedsensitivity of the iCycler may be due to longer reaction times (2 hoursv 1 hour) or larger sample volume (2 μl v 1 μl). The iCycler alsodisplays advantages in terms of throughput (96 v 32). There were nosamples reported positive using the iCycler and negative using theLightCycler. These results indicate that the LightCycler is bettersuited to specific analyses, whereas the iCycler is better suited tosensitive analyses.

Thirdly, there was one false positive observed with ScJC, which occurredin a control negative blank reaction. This observation highlights thesensitivity of the assay and the need for scrupulous assay technique andcaution when interpreting viral analyses of this sort. This problem hasbeen encountered before in laboratories responsible for testing UK poliovaccines with no convincing explanation as to the source ofcontamination. The fact that this false positive occurred in anexperiment that contained five other blanks, all producing the expectednegative result, suggests this was an isolated case. Also, the fact thatthe laboratory used has not been previously exposed to polyomavirusessuggests that this blank is positive as a consequence of being locatedadjacent to two positive controls. Thus, sample carryover may beresponsible.

The incidence of SV40 found has a maximum value of 70% and thus lieswithin, but towards the upper limit of, previously-reported ranges. JCand BK virus are estimated to occur in 60% of the paediatric populationwith over 70% of adults having JC and BK antibodies. An incidence of 33%has been reported for JC virus in tumours of the central nervous system.The incidence of JC and BK found was 19% and 1.4%, respectively.

Estimation of viral load in these samples is complicated by the use ofnested PCR in which reactions may reach ‘limiting conditions’ during thefirst round of amplification. SV40-VESPA is capable of typing cell lineDNA in a one round amplification (data not shown). Thus, those samplesthat are strongly positive in the nested screen could be re-analysed forviral load in a one round PCR using internal standards for human genomicDNA. The data show DNA samples with ScSV40 fluorescence in the range 1.4to 26.5, consistent with there being a large range of viral loadspresent in the mesothelium biopsies from which they were derived.

CONCLUSION

FIGS. 10 a-10 f demonstrate that the present invention results in anassay capable of producing strong signals in response to cognate virusand of discriminating cognate virus from large concentrations of highlyhomologous non-cognate virus. The one possible exception to thisstatement is the observation of significant background fluorescence whenScBK is challenged with pre-amplified JC virus. Although stillacceptable at around 10% of the positive control value, there is thepossibility of assigning JC virus as a false positive BK virus. Thisobservation does not affect the current study, since all samples weretyped for JC virus and the one sample positive for BK was JC negative.However, all BK positives produced using this technique must beinterpreted with caution and ideally typed for JC virus. It is worthre-emphasising, however, that ScBK background fluorescence is onlyobserved in the presence of pre-amplified JC DNA at high concentration.

Therefore, the central findings of this experiment are:

(i) The incidence of SV40 is dependent on size of amplicon probed;

(ii) The incidence of SV40 is dependent on the mode of ampliconanalysis; and

(iii) The rate of infection of SV40 in archival mesothelioma tissue isin the range of from 58% to 70%.

EXAMPLE 7 VESPA in HPV Associated with RRP

Clinical Samples

Patients were recruited by contacting all Ear, Nose and Throat surgeonsthroughout Wales and all those in England known to be actively engagedin recurrent respiratory papillomatosis (RRP) research. All cases ofRRP, as confirmed by characteristic histological changes on biopsyand/or those requiring more than 2 surgical interventions, wereidentified. Informed consent was obtained from all subjects. Biopsieswere collected, with informed consent, from patients undergoingtreatment for Recurrent Respiratory Papillomatosis at the UniversityHospitals of Wales, Cardiff and Sheffield.

Disease Severity

The grade of clinical disease was assigned using the parameters outlinedby Derkay et al in Laryngoscope 108(6) 935-7 (1998); this equationaccounts for both the number and time interval between surgeries.

DNA Purification

DNA was extracted from biopsies by incubating approx 1 mm³ of biopsymaterial in 1 ml 10 mM Tris hydrochloride pH7.4 containing 0.5 mg/mlproteinase K (Sigma, UK) for 1 hour at 56° C. 100 μl aliquots were thentaken and boiled for 10 minutes, cooled on ice for 5 minutes, spun at13,000 rpm for 3 minutes (in order to remove particulate matter) anddecanted into fresh tubes.

PCR EIA

In order to confirm HPV types, the reference technique of Jacobs et al(qv) for PCR-EIA was performed on all samples as described in Example 3.

Results

The viral loads presented in Table 10 were calculated as described inExample 2. Briefly, the ratio of the fluorescence signals produced usinga cognate HPV-self-probing amplicon and that of a self-probing amplicondesigned to detect a human genomic beta-globin housekeeping gene wascalculated.

Although there are clearly examples of patients with high viral load andmild disease (NL) and vice versa (DN), a patient who is in remission hadthe lowest viral load and the most severe patient had the highest viralload. This study suggests that the most significant prognostic marker inRRP is HPV-type; HPV-6 leads to mild and HPV-11 to aggressive disease.TABLE 10 Sample Viral Load Name HPV Type Onset Disease Grade Ratio MWneg (was 6) JORRP Regression −0.02 TW 6 JORRP Aggressive/Mild 1.15 JS 6JORRP Aggressive 1.34 DF 6 AORRP Mild 3.74 MP 6 AORRP Mild 4.25 RM 6JORRP Aggressive 4.63 DN 11 JORRP Aggressive 0.92 JJ 11 JORRP Aggressive2.41 NL 11 JORRP Mild 6.58 DH 11 JORRP Aggressive 24.12

1-55. (canceled)
 56. A self-probing amplicon comprising a nucleic acidsequence selected from the group consisting of: (i) SEQ IDs NOS: 1 to 9;(ii) SEQ ID NO: 11; (iii) SEQ ID NOS: 12 to 17; (iv) SEQ ID NOS: 18 to20; or (v) the group of self-probing amplicons for use in SV40, JC or BKdetermination consisting of ScSV40 primer, ScJC primer, ScBK primer, ScSV40 probe, Sc JC probe, ScBK probe, SV40 P1, SV40 P2, SV40 P3, SV40 P4,JC P1, JC P2, JC P3, JC P4, BK P1, BK P2, BK P3, or BK P4, and anymixture thereof.
 57. A self-probing amplicon according to claim 1,further including a tail region comprising SEQ ID NO:
 10. 58. Aself-probing amplicon according to claims 1 or 2, further including aprobe component adapted to bind to the GP6+ sequence.
 59. A self-probingamplicon comprising a nucleic acid sequence comprising: (a) a primercomponent; and (b) a probe component; wherein the primer componentcomprises a sequence selected from the group consisting of SEQ ID NOS:32 to 40, SEQ ID NOS: 47 to 52, or SEQ ID NO: 61; and wherein SEQ ID NO:61 is a primer component of SEQ ID NOS: 19-20.
 60. A self-probingamplicon comprising a nucleic acid sequence comprising: (a) a primercomponent; and (b) a probe component; wherein the probe componentcomprises a sequence selected from the group consisting of SEQ ID NOS:21 to 29, SEQ ID NO: 31, SEQ ID NOS: 41 to 46, or SEQ IDS NOS: 59-60;and wherein SEQ ID NOS:59-60 are the probe component of SEQ ID NOS:19-20.
 61. A diagnostic kit for the detection, typing, or determinationof viral cell load or viral integration state comprising: (a) at leastone virus self-probing amplicon, housekeeping self-probing amplicon, ortailed primer; and (b) instructions for use of the kit.
 62. A kit forestimating viral load per cell according to claim 6, comprising at leasttwo virus self-probing amplicons.
 63. A kit according to claim 6 orclaim 7, comprising at least one housekeeping self-probing amplicon.